Gel Electrophoresis Flashcards
Describe gel electrophoresis
The separation of charged molecules on the basis of size by sorting through a porous gel meshwork made of agarose and possible two polyacrylamide (used for proteins).
The shorter the fragment is, the faster it will travel because it moves through the gel more easily. Longer nucleotide fragments take longer to migrate.
What does gel electropherosis separate?
Desired genes cut from DNA into fragments from restriction enzymes. These fragments are of different lengths
Describe the procedure for gel electrophoresis
A solution containing different size fragments that are to be separated is put in a well or depression at one end of the gel. This DNA solution is mixed with a loading dye that allows DNA to be easily visualized.
Using a direct current (electricity), a negative charge is placed at one end of the gel and a positive charge is placed at the other end.
Negatively-charged DNA will migrate towards the positive charge depending on fragment size. Once complete, fragments are made visible by staining the gel with ethidium bromide (EtBr).
The bands that are produced by the fragments using specific restriction enzymes are characteristic for that particular DNA. Therefore, you can compare the DNA pattern that you obtain with a gel from other places. The greater the similarity in patterns between the two sources, the greater the chance of a match (applications: forensics, paternity, diseases, agriculture).
Review the diagram of gel electrophoresis
What are plasmids?
Small, circular, double-stranded DNA molecules lacking a protein coat. They naturally exist in the cytoplasm of many strains of bacteria. They are indépendant of the chromosome of the bacteria and range in size of 1000 to 2000 base pairs.
They often carry genes that express proteins able to confer antibiotic resistance.
How can we use plasmids for biotechnology?
Using restriction enzymes, we may be able to insert a gene, such as the gene for insulin, into a plasmid and have a bacteria replicate it and mass produce insulin for people that have Diabetes.
Describe the calcium chloride method of introducing plasmids into bacteria cells
Through transformation. Plasmids are small enough to easily introduce into a bacterial cell. The plasmid is used as the vector to carry the desired gene into a host cell.
If the bacterium accepts the foreign DNA, it is described as a competent cell.
Then, suspend the bacteria in a solution of CaCl2 (a salt that ionizes into Ca+2 and Cl-1) and bring the temperature to 0 deg. The positively charged Ca ions will stabilize the negative phosphates. The low temperature will freeze the membrane, making it rigid.
Next, the plasmid DNA will be introduced into the solution and the whole thing will be subjected to a heat shock treatment of 42 deg. C. that lasts for about 90 seconds. This creates a draft where the outside of the cell has a slightly higher temperature than the inside of the cell. The draft will cause the plasmid to be swept into the cell through the pores of the membrane.
Describe the modern method of introducing plasmids into bacteria cells
Electroporators are used. Chambers that subject the bacteria to an electric shock. The electric shock loosens the structure of the cell walls and allows foreign DNA to enter.
Plant cells are more difficult due to the cell walls. They have to use “gene guns” to “shoot” DNA through the cell wall and membrane.
Review the diagrams of plasmids
Describe PCR
Polymerase chain reaction. A direct method of making copies of a desired DNA sequence, a method alternative to inserting gene fragments into a plasmid.
It is closely related to the DNA replication method found in cells
Describe the process of PCR
DNA is separated using heat (94 - 96 deg. C.) to break the hydrogen bonds and the strands separate. Using primers and dropping the temperature to about 50-65 deg. C. allows the primers to anneal.
Once the primers have annealed, taq polymerase (isolated from Thermus aquaticus, a bacterium that lives in hot springs and therefore has enzymes that can withstand high temperatures), a DNA polymerase that builds complementary strands is done at 72 deg. C.
When the complementary strands are built, the cycle is then repeated. Each cycle doubles the number of double-stranded copies of the target DNA. After 30 days there are over a billion copies.
This method is often used in forensic science to amplify a small amount of DNA so that they can run gel electropherosis for DNA fingerprinting
Review the PCR diagram
Describe DNA fingerprinting
Restriction fragment length polymorphism. Polymorphism is an difference in DNA sequences that can be detected. They can be carried in coding regions which may be used to identify individuals with specific mutations. In non-coding regions, variations may exist between the number of VNTRs.
This process essentially involves the comparison of different lengths of DNA fragments produced by a restriction enzyme. It moves one step further than gel electropherosis.
